The invention relates to two novel laminins, i.e., laminin 13 and 14, and methods of making and using these molecules. The invention also relates to the use of laminin 5 to treat neural disorders, e.g., to induce or promote retinal adhesion and the viability of retina photoreceptors.
The laminins are large heterotrimeric glycoproteins of the extracellular matrix. Each laminin heterotrimer is composed of an xcex1, a xcex2, and a xcex3 chain, chosen from a number of possible homologues of each chain. Currently, eleven laminin chains have been identified: five xcex1 chains, three xcex2 chains, and three xcex3 chains (reviewed in [1]).
The invention is based, in part, on the discovery of two novel members of the laminin family, laminin 13 and laminin 14. Accordingly, the invention features a purified or isolated preparation, a recombinant preparation, or a composition of laminin 13, which includes laminin chains xcex13, xcex22, and xcex33. In a preferred embodiment, the laminin 13 is a trimer of an xcex13, xcex22, and xcex33 chain.
In a preferred embodiment, the xcex13 chain has a molecular weight of about 300 kD, 200 kD, or 165 kD, the xcex22 chain has a molecular weight of about 190 kD or 170 kD, the xcex33 chain has a molecular weight of about 200 kD or 170 kD.
In another preferred embodiment, the xcex13 chain is reactive or specifically binds to mouse monoclonal antibody BM-2 or any other antibody which can compete for the BM-2 epitope. In another preferred embodiment, the xcex22 chain is reactive or specifically binds to guinea pig polyclonal GP1 [47], mouse monoclonal C4 [46], R49, D5, D79, or any other antibody which can compete for the GP1 or C4 epitope.
In another aspect, the invention features, a purified or isolated preparation, a recombinant preparation, or a composition of laminin 14, which includes laminin chains xcex14, xcex22, and xcex33. In a preferred embodiment, the laminin 13 is a trimer of an xcex14, xcex22, and xcex33 chain.
In a preferred embodiment, the xcex14 chain has a molecular weight of about 185 kD, the xcex22 chain has a molecular weight of about 190 kD or 170 kD the xcex33 chain has a molecular weight of about 200 kD or 170 kD.
In another preferred embodiment, the xcex14 chain is renactive or specifically binds to a xcex14 rabbit polyclonal antibody disclosed in J. Cell Biol 1997, 137:685-701 or any other antibody which can compete for the epitope of the xcex14 rabbit polyclonal antibody. In another preferred embodiment, the xcex22 chain is reactive or specifically binds to guinea pig polyclonal GP1 [47], mouse monoclonal C4 [46], R49, D5, D79, or any other antibody which can compete for the GP1 or C4 epitope.
The laminin chains of any laminin as disclosed herein can be the initial translation product or a degradation product, e.g., a naturally occurring degradation product of a laminin chain.
In another aspect, the invention features a composition which includes a purified isolated or recombinant laminin 13, 14, or both. The invention includes pharmaceutical preparations, e.g., a pharamaceutical preparation which include a pharmaceutically acceptable carrier.
In another aspect, the invention features an isolated nucleic acid, e.g., DNA, RNA, or cDNA encoding laminin 13. The isolated nucleic acid can be a combination of nucleic acids each encoding one or more laminin chains or a single nucleic acid. The isolated nucleic acid can be expressed in a vector, e.g., an expression vector or expressed directly in a cell. A vector containing a sequence corresponding to the sequence of the isolated nucleic acid can express the isolated nucleic acid in a suitable cell or a suitable in vitro environment.
The invention also features an isolated nucleic acid, e.g., DNA, RNA, or cDNA encoding laminin 14. The isolated nucleic acid can be a combination of nucleic acids each encoding one or more laminin chains or a single nucleic acid. The isolated nucleic acid can be expressed in a vector, e.g., an expression vector or expressed directly in a cell. A vector containing a sequence corresponding to the sequence of the isolated nucleic acid can express the isolated nucleic acid in a suitable cell or a suitable in vitro environment.
In another aspect, the invention features a recombinant laminin 13 or laminin 14 which can be produced, e.g., by expressing the laminin chains of laminin 13 or laminin 14 in a suitable cell host and under a condition suitable for the laminin chains to form laminin 13 or laminin 14.
In a preferred embodiment, the laminin 13 differs from a naturally occurring laminin 13 at at least 1, but less than 5, 10, or 15 amino acid residues. In another embodiment, one, two, or each laminin chain of a laminin, differs from its naturally occurring counterpart at at least 1, but less than 5, 10, or 15 amino acid residues.
In a preferred embodiment, the laminin 14 differs from a naturally occurring laminin 14 at at least 1, but less than 5, 10, or 15 amino acid residues. In another embodiment, one, two, or each laminin chain of laminin 14, differs from its naturally occurring counterpart at at least 1, but less than 5, 10, or 15 residues.
In another aspect, the invention features, a method of isolating a laminin 13 or 14. The method includes:
providing retinal tissue, e.g., a tissue selected from the group consisting of retina interphotoreceptor matrix, retina outer plexiform layer, neural retina, Mxc3xcller cell, and a preparation of retinal neurons, and isolating the laminin 13, 14, or a preparation of both. The laminins can be isolated by the use of immuno affinity columns which use one or more mabs which are specific for the subunits of laminin 13 or 14.
In another aspect, the invention features, a method for producing laminin 13. The method includes:
providing recombinant nucleic acid which encodes a laminin xcex13 chain, a laminin xcex22 chain, and a laminin xcex33 chain, and expressing the nucleic acid to provide recombinant laminin 13.
In a preferred embodiment a single cell includes nucleic acid which encodes the laminin xcex13 chain, a laminin chain xcex22, and a laminin xcex33 chain.
In another aspect, the invention features, a method for producing laminin 14. The method includes:
providing recombinant nucleic acid which encodes a laminin xcex14 chain, a laminin xcex22 chain, and a laminin xcex33 chain, and expressing the nucleic acid to provide recombinant laminin 13.
In a preferred embodiment a single cell includes nucleic acid which encodes the laminin xcex14 chain, laminin xcex22 chain, and laminin xcex33 chain.
The invention still provides a method for treating a disorder associated with abnormal functions of synapses, e.g., insufficient stability, viability, formation, or defective organization of synapses. The method comprises administering to a subject an effective amount of laminin 13, laminin 14, laminin 5, separately or in combination with one another.
The invention provides a method for treating a disorder associated with inadequate neural cell growth, healing and regeneration,e.g., axon outgrowth, a disorder associated with abnormal subretinal space or interphotoreceptor matrix (IPM) such as inadequate stability of IPM, a disorder associated with retina contact, continuity, and/or adhesion, a disorder associated with abnormal or insufficient formation of synapses, and a disorder associated with viability of a neural cell, e.g., photoreceptor or an element thereof, e.g., outer segment, inner segment, cell body, and synapses. The method comprises administering to a subject an effective amount of laminin 13, laminin 14, laminin 5, separately or in combination with one another.
Still yet another feature of the present invention provides a method to treat a disorder associated with retinal abnormality, e.g., rod dystrophy, rod-cone dystrophy, macular degeneration, and retinal detachment. The method includes administering to a subject an effective amount of laminin 13, laminin 14, laminin 5, separately or in combination with one another.
Another feature of the present invention provides a method to induce neural cell growth or regeneration, e.g., axon outgrowth. The method includes administering to a subject an effective amount of laminin 13, laminin 14, laminin 5, alone or in combination with one another.
In a preferred embodiment, the method includes administering to a wound an effective amount of laminin 13, laminin 14, laminin 5, alone or in combination with one another.
Still another feature of the present invention provides a method to promote a condition, e.g., promote retina interphotoreceptor matrix stability, promote the stability of retina photoreceptor or an element thereof,e.g., outer segment, inner segment, cell body, and synapses, promote retina contact, continuity, and/or adhesion, promote the stability of synapses, and promote the formation of synapses. The method includes administering an effective amount of laminin 13, laminin 14, laminin 5, alone or in combination with one another.
Yet another feature of the present invention provides a method for preparing an implant, e.g., an implantable catheter, a retinal implant, a timed releasing device, a neural cell growth guide, an artificial tissue, an implant of the central nervous system, and an implant of the peripheral nervous system. The method includes contacting, e.g., coating or incubating the implant with laminin 5, 13, 14, alone or in combination with one another.
In a preferred embodiment, the implant is a subretinal implant, e.g., subretinal microphotodiodes, a visual prostheses, an implant for photoreceptor replacement, or a MPDA implant, e.g., as described in the abstract entitled xe2x80x9cCan Subretinal Microphotodiodes Successfully Replace Degenerated Photoreceptors?xe2x80x9d submitted by E. Zrenner et al at the Vision Research Conference held on May 9, 1998.
In another aspect, the invention features, a method of treating a disorder in a subject. The method includes: administering to the subject, an effective amount of laminin 13, laminin 14, or both.
In a preferred embodiment the disorder is: a disorder characterized by an insufficient level of a laminin, e.g., laminin 5, 13 or 14; a neural disorder; a disorder associated with neural tissue; disorder associated with abnormal functions of a synapse, e.g., insufficient stability, viability, formation, or the defective organization of a synapse; a disorder associated with inadequate neural cell growth, healing, or regeneration, e.g.,axon outgrowth; a disorder associated with abnormal subretinal space or interphotoreceptor matrix (IPM) such as inadequate stability of IPM; or a disorder associated with inadiquate viability of a neural cell, e.g., photoreceptor.
In a preferred embodiment the disorder is: a disorder associated with inadequate or insufficient contact, contunity, and/or adhesion between two structures, e.g., between a first and a second cell, e.g., a first and second neural cell, a first and second neural tissue, a first and second neural organ, e.g., brain and spinal cord, and a cell, e.g., a neural and a substrate, e.g., a membrane, and neural membranes or structures.
In a preferred embodiment the disorder is associated with a defect in retinal adhesion.
In a preferred embodiment the disorder is rod dystrophy, rod cone dystrophy, macular degeneration, or retinal detachment.
In preferred embodiments the laminin administered is laminin 13.
In preferred embodiments the laminin administered is laminin 14.
In another aspect, the invention features a method of treating a disorder in a subject. The method includes administering to said subject, an effective amount of laminin 5.
In a preferred embodiment the disorder is: a disorder characterized by an insufficient level of a laminin, e.g., laminin 5, 13, or 14; a neural disorder; a disorder associated with neural tissue; disorder associated with abnormal functions of a synapse, e.g., insufficient stability, viability, formation, or the defective organization of a synapse; a disorder associated with inadequate neural cell growth, healing, or regeneration, e.g., axon outgrowth; a disorder associated with abnormal subretinal space or interphotoreceptor matrix (IPM) such as inadequate stability of IPM; or a disorder associated with inadequate viability of a neural cell, e.g., photoreceptor.
In a preferred embodiment the disorder is: a disorder associated with inadequate or insufficient contact, contunity, and/or adhesion between two structures, e.g., between a first and a second cell, e.g., a first and second neural cell, a first and second neural tissue, a first and second neural organ, e.g., brain and spinal cord, and a cell, e.g., a neural and a substrate, e.g., a membrane, and neural membranes or.
In a preferred embodiment the disorder is associated with retina adhesion,
In a preferred embodiment the disorder is rod dystrophy, rod cone dystrophy, macular degeneration, or retinal detachment.
In another aspect, the invention features a method of increasing the stability of a biological structure. The method includes contacting the structure with an effective amount of laminin 13 or 14.
In preferred embodiments the method is performed: in vivo, e.g., on a human or animal subject; in vitro, e.g., on a cultured tissue or cell; ex vivo, e.g., on an tissue which will be implanted in a subject.
In preferred embodiments the structure comprises: the retina, or a component thereof, e.g., the retina interphotoreceptor matrix, a photoreceptor or an element thereof, e.g., outer segment, inner segment, cell body, and synapses; a neuron or synapse, or a tissue which includes a neuron or a synapse; a nerve fiber; and the spinal cord.
In another aspect, the invention features a method of increasing the stability of a synapse. The method includes contacting the synapse or a cell which forms the synapse with an effective amount of laminin 13, 14, or a combination thereof.
In a preferred embodiment, the synapse is a synapse of the central nervous system, or a synapse of the peripheral nervous system.
In preferred embodiments the laminin administered is laminin 13.
In preferred embodiments the laminin administered is laminin 14.
In another aspect, the invention features a method of increasing the stability of a biological structure. The method includes contacting the structure with an effective amount of laminin 5.
In preferred embodiments the method is performed: in vivo, e.g., on a human or animal subject; in vitro, e.g., on a cultured tissue or cell; ex vivo, e.g., on an tissue which will be implanted in a subject.
In preferred embodiments the structure comprises: the retina, or a component thereof, e.g., the retina interphotoreceptor matrix, a photoreceptor or an element thereof, e.g., outer segment, inner segment, cell body, and synapses; a neuron or synapse, or a tissue which includes a neuron or a synapse; a nerve fiber; the spinal cord.
In another aspect, the invention features a method of increasing the stability of a synapse. The method includes contacting the synapse or a cell which forms the synapse with an effective amount of laminin 5.
In a preferred embodiment, the synapse is a synapse of the central nervous system, or a synapse of the peripheral nervous system.
In another aspect, the invention features a method of promoting the contact, continuity, or adhesion of a first structure and a second structure. The method includes contacting at least one of the structures with an effective amount of laminin 13, 14, or both.
In a preferred embodiment: the first structure can be any of a cell, a membrane, a tissue, an organ, or a nerve fiber, and the second structure can be any of a cell, a membrane, a tissue, an organ, or a nerve fiber.
In a preferred embodiment, the first structure is a retinal cell and the second structure is a retinal cell.
In a preferred embodiment the first structure is a cell, e.g., a neural cell and the second structure is a substrate, e.g., a membrane.
In a preferred embodiment the first structure is a cell, tissue, or an organ, e.g., a neural cell, a nerve, brain, spinal cord, or a membrane and the second structure is a substrate, e.g., a surface of an implant, e.g., a prosethetic device, or an in vivo or ex vivo substrate, e.g., a substrate on which a cell or tissue is cultured.
In preferred embodiments the method is performed: in vivo, e.g., on a human or animal subject; in vitro, e.g., on a cultured tissue or cell; ex vivo, e.g., on an tissue which is implanted in a subject.
In preferred embodiments the structure comprises: the retina, or a component thereof, e.g., the retina interphotoreceptor matrix, a photoreceptor or an element thereof; a neuron or synapse, or a tissue which includes a neuron or a synapse; a nerve fiber; and the spinal cord.
In another aspect, the invention features a method of promoting retinal contact, continuity, or adhesion in a subject. The method includes administering an effective amount of laminin 13, 14, or both.
In a preferred embodiment the laminin is provided with a pharmaceutically acceptable carrier.
In a preferred embodiment an integrin is also administered.
In another aspect, the invention features a method for treating a subject having a retinal disorder, e.g., a disorder associated with insufficient retinal contact, continuity, and/or adhesion or retinal degeneration. The method includes administering to a subject an effective amount of laminin 13, 14 or both.
In a preferred embodiment, the disorder is: rod dystrophy, rod-cone dystrophy, macular degeneration, retinal detachment, or retinitis pigmentosa.
In preferred embodiments the laminin administered is: laminin 13.
In preferred embodiments the laminin administered is: laminin 14.
In another aspect, the invention features a method of stimulating the formation of a synapse. The method includes contacting the synapse, or a cell which forms the synapse with an effective amount of laminin 13, 14 or both.
In a preferred embodiment, the synapse is a synapse of the central nervous system, or a synapse of the peripheral nervous system.
In another aspect, the invention features a method of promoting the contact, continuity, or adhesion of a first structure and a second structure. The method includes contacting at least one of the structures with an effective amount of laminin 5.
In a preferred embodiment: the first structure can be any of a cell, a membrane, a tissue, an organ, or a nerve fiber, and the second structrue structure can be any of a cell, a membrane, a tissue, an organ, or a nerve fiber.
In a preferred embodiment, the first structure is a retinal cell and the second structure is a retinal cell.
In a preferred embodiment the first structure is a cell, e.g., a neural cell and the second structure is a substrate, e.g., a membrane.
In a preferred embodiment the first structure is a cell a tissue, or an organ, e.g., a neural cell, a neural tissue, brain, spinal cord, a nerve, or a membrane and the second structure is a substrate, e.g., a surface of an implant, e.g., a prosethetic device, or an in vivo or ex vivo substrate, e.g., a substrate on which a cell or tissue is cultured.
In preferred embodiments the method is performed: in vivo, e.g., on a human or animal subject; in vitro, e.g., on a cultured tissue or cell; ex vivo, e.g., on an tissue which is implanted in a subject.
In preferred embodiments the structure comprises: the retina, or a component thereof, e.g., the retina interphotoreceptor matrix, a photoreceptor; a neuron or synapse, or a tissue which includes a neuron or a synapse; a nerve fiber; and the spinal cord.
In another aspect, the invention features a method of promoting retinal contact, continuity, or adhesion in a subject. The method includes administering an effective amount of laminin 5.
In a preferred embodiment the laminin is provided with a pharmaceutically acceptable carrier.
In a preferred embodiment an integrin is also administered.
In another aspect, the invention features a method for treating a subject having a retinal disorder, e.g., a disorder associated with insufficient retinal contact, continuity, and/or adhesion or retinal degeneration. The method includes administering to a subject an effective amount of laminin 5.
In a preferred embodiment, the disorder is: rod dystrophy, rod-cone dystrophy, macular degeneration, retinal detachment, or retinitis pigmentosa.
In another aspect, the invention features a method of stimulating the formation of a synapse. The method includes contacting the synapse, or a cell which forms the synapse with an effective amount of laminin 5.
In a preferred embodiment, the synapse is a synapse of the central nervous system, or a synapse of the peripheral nervous system.
In another aspect, the invention features a method of increasing the viability of retina photoreceptors or a component thereof, e.g., outer segment, inner segment, cell body, and synapses. The method includes contacting the retina photoreceptors with an effective amount of one or more of laminin 5, 13, or 14.
In a preferred embodiment an integrin is also administered.
In another aspect, the invention features a method for promoting neural cell growth, healing, or regeneration, e.g., axon outgrowth. The method includes contacting the neural cell with an effective amount of one or more of laminin 13, 14, or 5.
In preferred embodiments the method is performed: in vivo, e.g., on a human or animal subject; in vitro, e.g., on a cultured tissue or cell; ex vivo, e.g., on an tissue which will be implanted in a subject.